Molecular Characterization and Human T-Cell Responses to a Member of a NovelMycobacterium tuberculosis mtb39Gene Family

Abstract

We have used expression screening of a genomic Mycobacterium tuberculosis library with tuberculosis (TB) patient sera to identify novel genes that may be used diagnostically or in the development of a TB vaccine. Using this strategy, we have cloned a novel gene, termed mtb39a, that encodes a 39-kDa protein. Molecular characterization revealed that mtb39a is a member of a family of three highly related genes that are conserved among strains of M. tuberculosis and Mycobacterium bovis BCG but not in other mycobacterial species tested. Immunoblot analysis demonstrated the presence of Mtb39A in M. tuberculosis lysate but not in culture filtrate proteins (CFP), indicating that it is not a secreted antigen. This conclusion is strengthened by the observation that a human T-cell clone specific for purified recombinant Mtb39A protein recognized autologous dendritic cells infected with TB or pulsed with purified protein derivative (PPD) but did not respond to M. tuberculosis CFP. Purified recombinant Mtb39A elicited strong T-cell proliferative and gamma interferon responses in peripheral blood mononuclear cells from 9 of 12 PPD-positive individuals tested, and overlapping peptides were used to identify a minimum of 10 distinct T-cell epitopes. Additionally, mice immunized with mtb39a DNA have shown increased protection from M. tuberculosis challenge, as indicated by a reduction of bacterial load. The human T-cell responses and initial animal studies provide support for further evaluation of this antigen as a possible component of a subunit vaccine for M. tuberculosis.

Expression Cloning of an Immunodominant Family of Mycobacterium tuberculosis Antigens Using Human Cd4+ T Cells

“…2. The DC-4 cell line showed strong reactivity with CFPs and rMtb39A 16 but not with any of the other recombinant antigens, whereas DC-6 reacted strongly with CFPs and Mtb11 and weakly with Mtb39A. In addition, DC-6 but not DC-4 reacted strongly with whole E .…”

Cell-Mediated Immune Responses to in vivo-Expressed and Stage-Specific Mycobacterium tuberculosis Antigens in Latent and Active Tuberculosis Across Different Age Groups

“…This rational has led to the identification of several TB vaccine candidates whose efficacy has been proven in animal models (55-57), but not yet tested in humans. It therefore remains to be seen whether there is a difference in efficacy between TB vaccine candidates based on antigens preferentially recognized by LTBI vs. TB patients (7,8,(58)(59)(60).…”

Expression Library Immunization: a Road Map for Discovery of Vaccines against Infectious Diseases

“…Only pools of immunogenic antigens were further screened in primates to identify protective vaccines. In another example using an M. tuberculosis genomic library expressed in Escherichia coli, only antigens recognized by rabbit antiserum directed against M. tuberculosis were further tested in a murine challenge system as vaccine candidates (28). Another modification of the main theme of ELI is to enrich the plasmid library for antigens that trigger a desired immune response before screening in animals.…”